PROJECT SUMMARY/ABSTRACT The goal of the proposed research is to develop breast cancer cell targeting peptides and peptide-drug conjugates for targeted delivery of chemotherapeutic agents specifically to breast cancer site. Chemotherapy is the treatment of choice for most cancers including the breast cancer. However, it is rarely curative and mostly ineffective due to intolerable toxicities of the chemotherapeutic agents. Chemotherapeutic agents, like doxorubicin and paclitaxel, are potent cytotoxic agents that enter healthy tissues in the body with indiscriminate toxicity and do not preferentially accumulate at tumor sites. One of the most effective strategies to improve the specific uptake of these agents to cancerous cells in tumors is using targeting ligands, such as engineered antibodies and tumor homing peptides, which bind specific receptors on cancer cells. We propose to develop peptide-drug conjugates (PDCs) where a chemotherapeutic drug doxorubicin will be conjugated to a breast cancer cell targeting bispecific peptide that facilitates delivery of the PDCs to triple-negative breast cancer (TNBC) cells sparing normal noncancerous cells. TNBC is an important subtype of breast cancer to study because it is more aggressive than other subtypes and chemotherapy is the mainstay of treatment for TNBC. The newly designed bispecific peptides consist of two peptides, peptide 18-4 (or GE11) and the pH dependent peptide pHLIP, that target cancer cells by two different mechanisms ensuring site specific delivery of the attached drug. Peptide 18-4 and GE11 target specific receptors, keratin 1 and epidermal growth factor receptor (EGFR), respectively, overexpressed on breast cancer cells, whereas peptide pHLIP targets breast tumors due to the low pH microenvironment. The bispecific peptides will be synthesized using solid phase peptide synthesis technique followed by conjugation via different linkers to doxorubicin to obtain PDCs. The PDCs will be evaluated for specific uptake by the TNBC cells, as well as, their toxicity toward TNBC cells will be determined. The anti-tumor efficacy of the PDCs will be assessed in breast cancer xenograft animal model. Evaluation using the in vitro and in vivo systems will ensure that our results are robust and reproducible, and the proposed conjugates succeed toward breast cancer treatment ultimately benefiting Women?s Health. Overall, the development of PDCs for improved efficacy of current chemotherapeutic drugs will be a key step forward in breast cancer treatment and will eventually help in the eradication of this growing disease.